Our research focuses on intrathymic T-cell development, which, as one of the few developmental programs that continue throughout life, is a privileged model to study cell death/survival decisions in mammals. After they have rearranged their T-cell Receptor (TCR) genes, immature thymocytes initiate the process known as T-cell selection, in which their fate, i.e. death vs. differentiation and survival, depends on signals generated by their TCR upon engagement by self-MHC peptide ligands. Our objective is to identify the intracellular signaling and gene expression programs involved in T-cell selection. We have been taking three concurrent experimental approaches to address these issues. First, we have generated mouse models to interrupt TCR signaling in vivo at defined steps of T-cell development. These mouse models were developed by restricting the developmental window of expression of Zap70, a tyrosine kinase required for TCR signal transduction. Using these models, we have shown that the duration of TCR signaling controls CD4-CD8 lineage choice in vivo and we have defined the developmental window during which thymocytes remain open to lineage choice. We have found that, contrary to expectations, late stages of thymocyte development (i.e. after the completion of lineage differentiation but before exit from the thymus) remain dependent on TCR signaling and we are currently investigating the targets of TCR signaling in such late stage thymocytes. Our second approach used high-throughput gene expression analyses to identify genes potentially involved in differentiation and survival during intrathymic selection. We have identified over a hundred genes whose expression is affected by selection signals, including genes with potential "regulatory" activity such as transcription factors or membrane receptors. After more detailed expression analyses, we have selected two promising "candidate" genes for further studies (currently in progress) using transgenic and homologous recombination approaches. The third strategy we are pursuing uses conditionally active proteins to control signaling in cell populations in a synchronous way in vivo. This approach, which involves the development of proteins with ligand-controled activity, has the advantage over "conventional" gene targeting or transgenesis to allow analyses of dynamic responses to perturbations in cells or organisms. Compared to ligand (e.g. tetracycline)-regulated expression systems, conditionally active proteins offer greater time-resolution by by-passing the time lag required for de novo gene expression to achieve sufficient protein concentrations. We have generated a conditional version of Zap70, whose activity can be controlled by small diffusible ligands, and we have validated this approach using transient expression in cell lines. Using this strategy, we have shown that Zap70 has a dual role in TCR internalization: recruitment of Zap70 without kinase activation protects TCR complexes from internalization, whereas kinase activation promotes TCR internalization. We are currently characterizing mouse transgenic lines expressing conditional Zap70 proteins, in order to study signal transduction and gene expression in vivo during intrathymic development.